Mitochondrial genome in sporadic breast cancer: A case control study and a proteomic analysis in a Sinhalese cohort from Sri Lanka

Breast cancer is the commonest malignancy in women and the majority occurs sporadically with no hereditary predisposition. However, sporadic breast cancer has been studied less intensively than the hereditary form and to date hardly any predictive biomarkers exist for the former. Furthermore, although mitochondrial DNA variants have been reported to be associated with breast cancer, findings have been inconsistent across populations. Thus we carried out a case control study on sporadic breast cancer patients and healthy controls of Sinhalese ethnicity (N = 60 matched pairs) in order to characterize coding region variants associated with the disease and to identify any potential biomarkers. Mitochondrial genome was fully sequenced in 30 pairs and selected regions were sequenced in the remaining 30 pairs. Several in-silico tools were used to assess functional significance of the variants observed. A number of variants were identified among the patients and the controls. Missense variants identified were either polymorphisms or rare variants. Their prevalence did not significantly differ between patients and the healthy controls (matched for age, body mass index and menopausal status). MT-CYB, MT-ATP6 and MT-ND2 genes showed a higher mutation rate. A higher proportion of pre-menopausal patients carried missense and pathogenic variants. Unique combinations of missense variants were seen within genes and these occurred mostly in MT-ATP6 and MT-CYB genes. Such unique combinations that occurred exclusively among the patients were common in obese patients. Mitochondrial DNA variants may have a role in breast carcinogenesis in obesity and pre-menopause. Molecular dynamic simulations suggested the mutants, G78S in MT-CO3 gene and T146A in MT-ATP6 gene are likely to be more stable than their wild type counterparts.

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Reviewer 1 -comments
Response The abstract must be made more crisp for better understanding with a graphical abstract which shapes the whole work.
Abstract has been modified to include the background to the study as requested by reviewer 2.
Graphical abstract has been included. The concluding segment of introduction is lagging with some more information's and the need of this work with a flow diagram.
The concluding segment in the introduction has been deleted as per suggestions of reviewer 2 and the objectives have been inserted. A flow diagram has been inserted as supplementary figure 1 and referred to in the text. Authors must take care of typo and grammar errors Corrected Authors must add a new section which displays the new findings in contraction of the old system and their new findings Using a case control study where breast cancer patients and healthy controls were matched for several confounding variables, we attempted to identify significant differences in the prevalence of coding region variants of the mt genome between the two groups. When compared with the previous studies in other populations, ours was a matched-pairs case control study which eliminated the effect of several confounding variables. Furthermore, it was limited to a single ethnic group to eliminate the effect of ethnicity on the mtDNA variants. We also stratified our analysis by menopausal status, tumour histology and body size to unravel any differences in the outcome and observed a greater association of mtDNA variants with breast cancer in pre-menopausal women and in obese women. Several in-silico tools were used for assessing functional significance of the variants and selected variants were subjected to Molecular Dynamic simulations. To our knowledge, many studies which previously reported association of mitochondrial variants with breast cancer did not assess functional significance of the variants and the few which did so used a fewer number of in-silico tools. Molecular Dynamic simulation of identified variants does not seem to have been carried out in previous studies.

Reviewer 2 -comments
Response In DNA extraction method, what does it mean by "..from the remainder"? Explain the remainder. In MD simulation "….using an NVT ensemble for 100 ns" is quite long. Generally for 10-15 ns is enough. Recheck Replaced "remainder" with "remaining 20 samples for clarity". It was run for 10ns, not 100ns. It was corrected in the manuscript What is the exact production run time in MD simulation?

ns
Only 20 ns run is quite impossible to suggest stability of a mutant protein. In my opinion at least 200 ns or more simulation time will depict the clear picture.
In view of the practical difficulty of simulating large protein complexes with the membrane environment, Short 20 ns simulations were carried out to capture mainly the local motions and essential collective dynamics of the mutant and wild type proteins (Karami et al, 2018). Dynamic simulations of 20ns (Dorosh et al, 2013, Zhang et al, 2017, Mercer et al, 2018, Muneeswaran et al, 2018 or even less (Blinov et al, 2009) has been applied in previous studies for similar predictions and even to reproduce some experimental evidence of the stability of mutant proteins in silico (Zhang et al, 2017) Added the phrase likely to be in the abstract (last line) and discussion (9 th para) when referring to stability of the mutant proteins. Explain the background of the study in the abstract. Abstract is too brief.
"Mitochondrial genome was analysed in sporadic breast cancer patients of Sinhalese ethnicity" has been replaced by "Breast cancer is the commonest malignancy in women and the majority occurs sporadically with no hereditary predisposition. However, sporadic breast cancer has been studied less well than the hereditary form and to date hardly any predictive biomarkers exist for the former. Furthermore, though mitochondrial DNA variants have been reported to be associated with breast cancer, findings have been inconsistent across populations. Thus we carried out a case control study on sporadic breast cancer patients and healthy controls of Sinhalese ethnicity (N=60 matched pairs) in order to characterize coding region variants associated with the disease and to identify any potential biomarkers. Mitochondrial genome was fully sequenced in 30 pairs and selected regions were sequenced in the remaining 30 pairs. Several in-silico tools were used to assess functional significance of the variants observed. A number of variants were identified among the patients and the controls." Highlight the scope of the study in the conclusion section preferably in a single paragraph.
Inserted "In view of the potential of using mtDNA variants to predict sporadic breast cancer, inconsistencies regarding association of specific mtDNA variants with breast cancer across populations and lack of such data for Sri Lankans, we analysed the mt genome in sporadic breast cancer of Sinhalese women and assessed the functional significance of the variants identified in-silico." Indicate exclusively the objectives of the study in the last paragraph of the introduction. Concluding remarks should be removed. So delete the sentences "Although many germ line variants were Deleted "Although many germ line variants were unique to breast cancer they were mostly confined to one or very few individuals. Some mutants appeared to be more stable than their wild type counterparts" unique to breast cancer they were mostly confined to one or very few individuals. Some mutants appeared to be more stable than their wild type counterparts".
Inserted "Using a matched-pairs study design coding region of the mitochondrial genome was compared between sporadic breast cancer patients and healthy controls matched for age, body mass index and menopausal status. Prevalence of identified variants were compared between the two groups and subsets based on the histology of the tumour and body size. Rate of mutation was computed for each mitochondrial gene. Variants identified were tested in-silico for functional significance using several tools and selected variants were further analysed using Molecular Dynamic simulations. The work is well-written, although there are several acronyms scattered throughout. It is encouraged that writers use complete words rather than abbreviations for greater clarity.
Several acronyms which appeared three times or less were removed and full name used. Other acronyms which were kept are common acronyms such as DNA, NGS, PCR etc. or names of genes, tools, Databases. Two acronyms in relation to histopathological diagnosis was kept as they are within parenthesis as these are commonly used abbreviations in histopathology. A supplementary file giving all the acronyms has been included as S8 Table.